The commercial production of mushrooms (Agaricus bisporus) involves a series of steps, including compost preparation, compost pasteurization, inoculating the compost with the mushroom fungus (spawning), incubation to allow thorough colonization of the compost with mushroom mycelia, top dressing the compost with moistened peat moss (casing), and controlling the environment to promote the development of mature mushrooms. The mushroom growing process is described in detail in several publications (for example, Chang & Hayes, 1978; Flegg et al., 1985; Chang & Miles, 1989; Van Griensven, 1988).
Mushroom production proceeds via a sequence of steps. First, the nutritive medium (compost) is inoculated with mushroom mycelia by distributing particulate material colonized with mushroom mycelia (spawn) through it, and then the compost is colonized with the mycelium. Mushroom spawn is generally made with sterilized grain that is inoculated with pure cultures of the desired mushroom strain. Virtually all spawn used to inoculate mushroom compost is made using rye, millet, wheat, sorghum, or other grain substrate. Next, the colonized compost is covered by a layer of nutrient poor material (casing). The casing layer is usually composed of moistened peat moss and limestone. The mycelium proceeds to colonize this layer, and once the casing layer is colonized, the growing room environment is altered to promote the formation of fruiting bodies. These fruiting bodies are harvested and sold as commercial mushrooms.
The time-consuming stages of mushroom production are the periods when the mycelia are colonizing a new material. One way that has been used to accelerate the process is to mix colonized material, such as colonized compost, into the casing layer on top of the compost in the bed. Overall production time is reduced, because growth of the mycelia into the casing starts at many points throughout the casing layer rather than just at the interface with the colonized compost. By including a small amount of colonized material in the nutrient poor casing layer, the casing layer is colonized more quickly and the onset of mushroom production occurs earlier. This effectively increases the annual capacity of a mushroom farm without capital investment. This procedure is commonly referred to as "CACing" (Compost At Casing).
Although the CACing procedure accelerates production and increases capacity for a fixed area of mushroom bed, the colonized compost that is added to the casing layer as inoculum must come from somewhere. If it comes from mushroom beds, capacity equal to the amount of bed volume used to provide the casing inoculum is lost. Another problem with using locally developed inoculum is controlling quality. If the compost is weakly colonized, then the CACing procedure will not increase the rate of colonization enough to compensate for the loss of bed capacity. Even worse, because this supplemental inoculum is not sterilized, it has the potential for spreading infection by contaminating microorganisms throughout the mushroom house, thereby reducing the productive capacity of the house.
Some mushroom farmers have attempted to solve these problems by inoculating the casing layer with the same spawn that is used to inoculate the compost layer. Spawn for inoculating the compost layer is produced by aseptically mixing pure mushroom mycelia with sterile grains and incubating to allow colonization of the grain. The grain spawn has a consistent level of live mycelial content, but has a relatively high nutrient content (contributed by the grain). In order to be effective, the inoculum must be supplied at relatively high levels, and the resultant nutrient level in the casing layer can inhibit the formation of the mushroom primordia (fruiting bodies). It also increases the potential for growth of contaminating organisms (especially molds) by providing them with nutrients that are normally absent from the casing layer.
Recognizing these problems, Romaine (U.S. Pat. No. 4,803,800) teaches the production of a mushroom synthetic CACing agent (i.e., casing spawn) by encapsulation of nutrients in a hydrogel polymer. The sterilized substrate is inoculated with pure cultures of the mushroom fungus and inoculated in a manner equivalent to grain spawn, resulting in a consistent level of live mycelial content. The synthetic CACing agent is used to inoculate the mushroom casing layer rather than the compost. Use of this synthetic CACing agent speeds fruiting in the same manner as the natural CACing with compost. Nitrogen contents in the Romaine synthetic CACing agent are generally low, which helps to reduce the growth of competitor microorganisms. For example, Romaine teaches total nutrient levels of 2 to 6% (wt/vol of formula). Assuming the use of 100% protein as the nutrient source, total nitrogen would be about 0.96%. Some of Romaine's formulas contain Perlite, vermiculite, soy grits, or similar materials at about 2 to 6% (wt/vol) of the formula as texturizing agents.
Dahlberg & LaPolt (U.S. Pat. No. 5,503,647) teach the development of a mushroom casing spawn prepared from nutritionally inert particles (calcined earth, vermiculite, Perlite, etc) amended with nutrients. The casing spawn is formulated with low nitrogen contents (generally less than 1%) to allow inoculation of the mushroom casing layer with Agaricus bisporus mycelium without promoting the growth of pests and pathogens. Dahlberg & LaPolt teach that high levels of proteinaceous ingredients such as soybean fines, etc. are inhibitory to Agaricus bisporus growth. Generally, nitrogen levels above about 2% in a casing spawn formula result in reduced growth of Agaricus bisporus mycelium. This casing spawn formulation is also proposed as a substrate for inoculation of spawn during its preparation.
A number of "synthetic" or "non-grain" spawns have been taught. Stoller (U.S. Pat. No. 3,828,470) teaches spawn for use in inoculating compost in which the cereal substrate has been diluted with an inorganic material containing calcium carbonate or an organic flocculating agent. Stoller also teaches that mushroom mycelium will not grow on feedstuffs such as cottonseed meal, soybean meal, etc., when used alone as an autoclaved substrate. Nitrogen contents in Stoller's examples are generally low. For example, Stoller's example 16 is estimated to contain about 0.22% nitrogen. Stoller's example 18 is estimated to contain about 0.7% nitrogen. Stoller also teaches that a fine, granular or powdery spawn is preferable to the large, whole grain particles of grain spawn. This is generally due to the number of "points of inoculum" per unit weight of spawn. While there is no evidence that any of Stoller's formulations have ever been used as casing spawn, there is no known reason why they would not be satisfactory for this purpose.
Fritsche (1978) describes a formula reported by Lemke (1971) for spawn on a perlite substrate. The formula is as follows: perlite (1450 g), wheat bran (1650 g), CaSO.sub.4.2 H.sub.2 O (200 g), CaCO.sub.3 (50 g), water (6650 ml). The pH after sterilization is 6.2 to 6.4. This formula is calculated to contain 1.10 to 1.34% nitrogen on a dry weight basis (assuming a typical nitrogen content of wheat bran of 2.24 to 2.72%). While there is no evidence that this perlite spawn formula has ever been used as casing spawn, there is no known reason why it would not be satisfactory for this purpose. Brini & Sartor (European Patent Application No. EP 0 700 884 A1) teach a substrate for inoculating mushroom compost consisting of a water retaining-dispersing agent (e.g. peat), a buffer, a protein containing component (e.g. soybean meal), a growth promoting material (e.g. corn gluten and/or corn starch) and water. The mixture is sterilized, inoculated with the mushroom fungus, and used to spawn mushroom compost. Nitrogen contents of the mixtures are 1.4 to 8.0 wt % nitrogen (based on the specified range of 4 to 20% protein). One would not expect this formulation to be successful as a casing spawn due to its high nitrogen content and the inhibitory effect of high nitrogen contents in mushroom casing layers (U.S. Pat. No. 5,503,647).
Several spawn makers have developed casing spawn products. While specific formulations are proprietary, most appear to be combinations of peat moss, vermiculite, perlite, charcoal, shredded compost, or other proprietary ingredients supplemented with low levels of nutrients. Table 1 summarizes nitrogen contents of eight commercially available casing spawn products. The analyses of the casing spawns were performed by the present inventors. Seven of the products have nitrogen contents at or below 1.2%, while one product has a nitrogen content of 2.01%. The low nitrogen contents of commercially successful casing spawn formulations confirms the widely held and stated (U.S. Pat. No. 5,503,647) beliefs that high nitrogen contents in the mushroom casing layer are detrimental to mushroom production.
TABLE 1 ______________________________________ Nitrogen contents of commercially available casing spawn formulations. CASING SPAWN MANUFACTURER WT % NIIROGEN ______________________________________ AMYCEL SPAWN COMPANY 1.17 LAMBERT SPAWN COMPANY 2.01 Le LION SPAWN COMPANY 1.16 INTERNATIONAL SPAWN LABORATORY 0.69 ITAL SPAWN 1.19 SYLVAN SPAWN COMPANY 0.58 SWAYNE SPAWN COMPANY 1.13 VLASIC FARMS, INC 1.20 ______________________________________
The mushroom science literature contains several references to the detrimental effects of high nutrient levels, especially nitrogen, in the casing layer. U.S. Pat. No. 5,503,647 specifically states that casing spawn formulas containing greater than 0.7% bioavailable Kjeldahl nitrogen result in reduced mushroom yields.
It is important to distinguish available nutrients from non-available nutrients. Sphagnum peat moss frequently used in mushroom casing layers may contain from 0.75 to 3.5% Kjeldahl nitrogen (Fuchsman, 1986). Hypnum, or "black" peat, contains significantly higher nitrogen levels. Both of these peats, however, support good fruiting of Agaricus bisporus when used as a casing material. The nitrogenous materials in peats are generally "humic" in character, such as lignin and other mineralized nutrients. These are not readily available to the mushroom fungus and most microorganisms capable of colonizing the casing layer. If additional nutritionally available nitrogen compounds are added to the casing layer, fruiting is inhibited.
The single known exception to the paradigm of the inhibitory effect of available nitrogen in the casing layer is a report by Nair et al. (1993). These authors treated cottonseed meal with either formaldehyde or calcium sulfate to prepare a delayed release supplement similar to the technology used for compost supplements (i.e, see U.S. Pat. No.3,942,969). The supplements were added to the mushroom casing layer at up to 8% of the fresh weight of the mixture. Mushroom yield increases of between 0.5% and 52% were attributed to this casing layer supplementation. The authors speculate that the preservative treatments denatured the proteins in the cottonseed meal and made them unavailable to competing microorganisms. They also considered the possibility that residual formaldehyde could suppress the growth of microbial competitors.
While Nair et al. (1993) reported that addition of treated cottonseed meal to the casing mixture did not result in the growth of fungal contaminants, tests in the present inventors' laboratory showed that this supplementation strategy often fails due to the heavy growth of molds. Further, the use of formaldehyde as a preservative treatment could represent a health and safety hazard. To the inventors' knowledge, the supplementation strategy described by Nair et al. (1993) has not been commercialized successfully.